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Fusion of vesicles with the air–water interface: the influence of polar head group, salt concentration, and vesicle size
Biochimica et Biophysica Acta 1463 (2000) 301^306 www.elsevier.com/locate/bba
Fusion of vesicles with the air^water interface: the in£uence of polar head group, salt concentration, and vesicle size M. Gugliotti *, H. Chaimovich, M.J. Politi Departamento de Bioqu|¨mica, Instituto de Qu|¨mica, Universidade de Sa¬o Paulo, Av. Prof. Lineu Prestes 748, 05508-940 Sa¬o Paulo, Brazil Received 24 August 1999; received in revised form 12 October 1999; accepted 19 October 1999
Abstract Fusion of vesicles with the air^water interface and consequent monolayer formation has been studied as a function of temperature. Unilamellar vesicles of DMPC, DPPC, and DODAX (X = Cl3 , Br3 ) were injected into a subphase containing NaCl, and the surface pressure (tension) was recorded on a Langmuir Balance (Tensiometer) using the Wilhelmy plate (Ring) method. For the zwitterionic vesicles, plots of the initial surface pressure increase rate (surface tension decrease rate) as a function of temperature show a peak at the phase transition temperature (Tm ) of the vesicles, whereas for ionic ones they show a sharp rise. At high concentrations of NaCl, ionic DODA(Cl) vesicles seem to behave like zwitterionic ones, and the rate of fusion is higher at the Tm . The influence of size was studied comparing large DODA(Cl) vesicles with small sonicated ones, and no significant changes were found regarding the rate of fusion with the air^water interface. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Vesicles; Phase transition temperature; Surface pressure; Fusion; Interface
1. Introduction Vesicles and liposomes may disrupt upon contact with the air^water interface forming a monolayer [1^ 3]. As a consequence of monolayer formation, the surface pressure, Z (or tension, Q) of a vesicle dispersion changes. This phenomenon has been detected in dispersions of vesicles and liposomes made of di¡erent types of lipids, natural and synthetic ones [4,5], and kinetic studies [6^8] have been developed to characterize the process. Fusion (or spreading) of vesicles at the surface of solutions has been used as an alternative to form monolayers with attached pro-
* Corresponding author. Fax: +55-11-815-5579; E-mail: [email protected]
teins [9,10]. Most of the works are focused on the in£uence of temperature on the rate of vesicles fusion with the air^water interface, which has various implications of biological and pharmacological interest, since the transformation of a closed bilayer into a monolayer is an alternative to study the problem of the lung surfactant [11,12]. Fusion of vesicles with the air^water interface is a spontaneous process, and the large in£uence of the phase transition temperature of vesicles (Tm ) [13] on the rate of fusion has been noticed. Fusion becomes much faster as the temperature of the system approaches the Tm [13^15]. The correlation between temperature and rate of fusion leads to the possibility of studying bilayers properties like permeability and interbilayer fusion using this phenomenon, since these properties depend also strongly on the temper-
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ature and are related to structural changes on the membranes. Recently, we showed that is possible to determine the Tm of vesicles using this methodology [16,17]. The aim of the present work is to study the di¡erences between ionic and zwitterionic vesicles regarding the rate of fusion with the air^water interface below and above the Tm . From the results is also possible to estimate de Tm of the vesicles. DODA(Cl) vesicles were chosen to verify the in£uence of salt concentration and size. 2. Materials and methods 2.1. Materials Dioctadecyldimethylammonium chloride and bromide (DODA(Cl)/DODA(Br)) were purchased from Kodak, and recrystallized using methanol/acetone 5:95 (v/v). NaCl (Merck) was of analytical grade. Dimyristoyl/dipalmytoylphosphatidylcholine (DMPC/DPPC) were purchased from Sigma. DPPC and DMPC were analyzed by thin-layer chromatography and showed just one spot. The water used in all experiments was doubly distilled and ¢ltered using a Milli-Q (Millipore) apparatus. 2.2. Methods 2.2.1. Vesicle preparation and properties The concentrations of all vesicle dispersions used are expressed in terms of the lipid monomer concentrations. Di¡erent volumes of small unilamellar vesicles dispersions were prepared by sonicating (15 min) lipid suspensions (4 and 10 mM) at about 10³C above the Tm of the vesicles using the titanium tip sonicator Braunsonic 1510 (B. Braun). The NaCl solutions for sonication were of the same concentration of those used for the surface pressure (tension) assays, in order to ensure an isosmotic medium. After sonication, the vesicle dispersions were centrifuged (10 000Ug for 20 min) in order to remove the titanium particles. Vesicle size was determined by dynamic light scattering (He^Ne laser, 60 mW, EMI photomultiplier, Brookhaven BI90 autocorrelator). Vesicle hydrodynamic diameters were 360 þ 50, î for DODA(Cl), 400 þ 50, 450 þ 50, and 570 þ 50 A DODA(Br), DMPC, and DPPC, respectively. Large
unilamellar DODA(Cl) vesicles were prepared by the injection method [18] yielding vesicles with a diameî . The NaCl concentration for the ter of 3800 þ 50 A light-scattering assays was 1 mM for all types of vesicles. 2.2.2. Surface pressure (Z) and surface tension (Q) measurements The Te£on trough consisted of a hollow circular compartment (diameter 6 cm) connected to a water bath circulator system. The temperature was measured inside the trough using a Te£on-coated thermometer (0.5³C accuracy). Vesicle aliquots were injected into the subphase using a glass pipette, followed by magnetic stirring using a Te£on stir bar. Z was measured by the Wilhelmy plate method [19] on a Langmuir balance (KSV Instruments, model KSV 5000), placed in a clean room. The same procedure was carried out using the Ring method [20], and Q was measured on a Du Nouy tensiometer (Fisher Scienti¢c Tensiomat, Model 21, Fisher Scienti¢c). 2.3. Experimental procedure The assays were carried out in four steps. (1) An aliquot of the vesicle dispersion (1, 3, 5 ml,T) was injected into the subphase. The system was stirred very carefully during the next 10 s. Following this, the surface was cleaned by aspiration using a Te£on tube connected to a vacuum pump. After setting Z to zero, the assay began at the selected temperature; (2) Z was recorded continuously as a function of time; (3) after the measurement, the temperature was set to the new value; (4) when the system reached the new temperature, the surface was cleaned by aspiration and the assay restarted without further stirring. The same procedure was carried out when using the tensiometer, but in this case Q was measured every 5 min. 3. Results 3.1. In£uence of polar head group Fig. 1 shows the results obtained using the Langmuir balance for DODA(Br) and DPPC sonicated vesicles. The surface pressure Z was recorded at sev-
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Fig. 1. (A) vZ/vt as a function of temperature for 5 ml of 5 mM of small DODA(Br) vesicles injected into 35 ml of a 1 mM NaCl subphase. (B) Ten ml of 5 mM of small DPPC vesicles were injected into 30 ml of a 1 mM NaCl subphase. The insets show Z recorded at several temperatures on a Langmuir balance.
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against the temperature, and the same result was found for DODA(Br) vesicles (Fig. 2). For DPPC and DMPC vesicles, vQ/vt showed a peak at 40³C and 25³C, respectively (Fig. 2), which are the Tm for these vesicles. 3.2. In£uence of vesicle size
Fig. 2. vQ/vt as a function of temperature for 7 ml of 5 mM of small DODA(Br) vesicles (F), 5 ml of 2 mM of small DPPC vesicles (a), and 5 ml of 5 mM of small DMPC vesicles (R). The subphases consisted of 40 ml of a 1 mM NaCl solution.
eral temperatures (Fig. 1, insets), and the tangents to the curves taking into account the ¢rst minute (vZ/ vt, where Z is the surface pressure and t is time) were calculated using KSV-LB5000 software, and plotted as a function of temperature. For DODA(Br) vesicles (Fig. 1A) it is possible to note a sharp rise in vZ/vt after 35³C, which corresponds to the Tm of these vesicles. A di¡erent pattern is observed for DPPC vesicles (Fig. 1B), where vZ/vt reaches a maximum at 39³C, and decreases thereafter. The same assay was carried out using the tensiometer for DODA(Br), DPPC, and DMPC sonicated vesicles. The surface tension decrease rate vQ/vt was plotted
Fig. 3. vZ/vt as a function of temperature for 5 ml of 5 mM of small DODA(Cl) vesicles (a), and for 10 ml of 10 mM of large DODA(Cl) vesicles (F) injected into 35 ml of a 1 mM NaCl solution.
Fig. 3 compares the results for small and large unilamellar DODA(Cl) vesicles. From the results obtained using the Langmuir balance, vZ/vt was calculated and plotted against the temperature. The NaCl concentration of the subphase (1 mM) was the same for both types of vesicles. A sharp rise was found at 36³C and 37.5³C for the small and large DODA(Cl) vesicles, respectively. 3.3. In£uence of salt concentration Fig. 4 shows the results of small DODA(Cl) vesicles prepared in 10 mM and 20 mM NaCl solutions. The change on the behavior of the fusion rate was observed in the 10 mM NaCl solution, and for 20 mM of NaCl, the vZ/vt pattern has changed completely. For these two cases, a peak at 40³C determines the Tm , a few degrees higher than at lower concentrations of NaCl. The same result was obtained using a 50 mM NaCl solution as subphase (data not shown).
Fig. 4. vZ/vt as a function of temperature for 10 ml of 5 mM of small DODA(Cl) vesicles injected into 30 ml of NaCl, 10 mM (a) and 20 mM (F).
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Table 1 Comparison between the transition phase temperatures obtained using the tensiometer and the Langmuir balance with the literature values for Tm of sonicated vesicles Vesicles
Tensiometer (³C)
Langmuir balance (³C)
Literature value (³C)
Refs.
DODA(Cl) DMPC DPPC DODA(Br)
^ 25 40 35
36 ^ 39 35
37.5 22^29 41 36^40a
[17,18,28] [29]b [30,31] [31^33]
a
Di¡erent values between 36³C and 40³C are found for DODA(Br) in the literature. Ref. [29] is a database containing 702 records on the Tm of DMPC vesicles. The most common temperature found is 24³C. b
4. Discussion The biggest advantage of this method is that the rate of fusion does not depend on the concentration of the vesicles when high-concentration samples are used [12,14]. According to the results obtained by Pattus and Schindler [2,3], 75% of the vesicles remain like layers directly beneath the water surface in equilibrium with those in the bulk. This equilibrium is governed by the di¡usion of the vesicles to the bulk, which changes when low-concentration samples are used. In addition, the equilibrium between the vesicles is a¡ected during the assay by aspirating the surface, because part of the vesicle population may be lost, decreasing the concentration. Some authors have reported changes on vZ/vt when very low concentrations of vesicles are used [21,22]. Besides, even where using high concentrations of vesicles (5, 10 mM) to avoid this, £uctuations may occur because of the raising and lowering of the ring, when using the tensiometer, due to the change in the equilibrium between the vesicles near the interface and those in the bulk. Thus, for a simple veri¢cation, all assays were repeated at least two times for all types of vesicles, and with several di¡erent total vesicle concentrations, no change in the results was found. A more accurate determination of the Tm by this methodology is possible using lower concentrations of vesicle dispersions. In this case, Z or Q are recorded continuously as a function of temperature, without any interference at the surface (aspiration) during the assay [16,17]. However, in this approach is not possible to detect di¡erences between ionic and zwitterionic vesicles. The in£uence of the polar head group is clearly observed in Figs. 1 and 2. It is interesting to note that, for zwitterionic vesicles, plots
of vZ/vt (or vQ/vt) show similar results to those obtained from permeability studies [23,24], where the maximum in permeability occurs when both the gel and liquid^crystalline phases coexist. The results presented here indicate that the zwitterionic vesicles are relatively stable below and even above the Tm , and at this temperature they fuse with the interface easily. On the other hand, the ionic vesicles showed a continuous increase in the rate of fusion above the Tm . In fact, an increase in permeability of DODA(Cl) vesicles at temperatures above the Tm has been reported [25]. It is known that DODA(X) (X = Cl3 , Br3 ) vesicles are not stable in the presence of high concentrations of salt [26]. Vesicle dispersions prepared with 10 mM of NaCl did not show high turbidity, but the samples prepared with 20 and 50 mM of NaCl were very turbid. However, the addition of salt from 10 up to 50 mM showed the same change on the fusion rate of DODA(Cl) vesicles, that is, a peak instead a sharp rise at the Tm , with a slight increase on its value. This e¡ect is assigned to the lowering of the degree of ionization of the vesicles as a function of the increase in salt concentration in the subphase. As the ionic strength increases, the repulsion between the charged head groups decreases, allowing the lipids to stay closer to each other [27]. This represents a gain in stability, and it is seen as an increase on the Tm . No signi¢cant changes were found between the rate of fusion with the interface for large and small DODA(Cl) vesicles, although di¡erent results are found in the literature [3,6]. It is possible to observe that the sharp rise on vZ/vt occurs at 36³C and 37.5³C for the small and large DODA(Cl) vesicles, respectively, which corresponds to the Tm region for these vesicles.
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Table 1 summarizes the results for Tm determination obtained using the tensiometer and the Langmuir balance for each type of vesicle, and the literature values for Tm are also listed for comparison. The Tm values found by analyzing the rate of fusion are in good agreement with those obtained by other techniques (see references in Table 1). 5. Conclusions The Tm values obtained using the tensiometer and the Langmuir balance are nearly the same, and the results are in good agreement with those obtained by other methods. A di¡erence in the behavior between zwitterionic and ionic vesicles at low salt concentration is observed. The results show that the ionic vesicles continue to fuse with the air^water interface above their Tm , while for the zwitterionic ones the maximum rate of fusion occurs at this temperature. The addition of salt to the ionic vesicles changed their behavior into that of zwitterionic ones. Although DODAX (Cl3 , Br3 ) vesicles do not present good stability in the presence of salt, it was possible to detect the change on the rate of fusion with the air^water interface at the Tm , and a change on its value due to high salt concentrations of NaCl. No signi¢cant changes on the Tm values were found between small and large DODA(Cl) vesicles, since the di¡erence of 1.5³C is within experimental error. These results strongly suggest that the mechanism of fusion with the interface for DODA(Cl) vesicles does not depend on the curvature radii of these bilayers. Acknowledgements The authors wish to thank Fabio Florenzano for the light-scattering measurements and Prof. Dr. Iolanda M. Cuccovia for preparing the injected vesicles. This work was supported by the following Brazilian ¢nancial agencies: CNPq, FAPESP, FINEP, and CAPES. References [1] R. Verger, F. Pattus, Chem. Phys. Lipids 16 (1976) 285^291.
[2] F. Pattus, P. Desnuelle, R. Verger, Biochim. Biophys. Acta 507 (1978) 62^70. [3] H. Schindler, Biochim. Biophys. Acta 555 (1979) 316^336. [4] T. Ivanova et al., Prog. Colloid Polym. Sci. 79 (1989) 24^32. [5] R. Qiu, R.C. MacDonald, Biochim. Biophys. Acta 1191 (1994) 343^353. [6] B.A. Denizot et al., J. Colloid Surf. Sci. 143 (1991) 120^126. [7] V. Raneva, Tz. Ivanova, G. Lazarova, J. Proust, I. Panaiotov, Colloid Polym. Sci. 273 (1995) 150^155. [8] C.S. Vassilie¡, I. Panaiotov, E.D. Manev, J.E. Proust, Tz. Ivanova, Biophys. Chem. 58 (1996) 97^107. [9] H. Schindler, Th. Schurholz, Eur. Biophys. J. 20 (1991) 71^ 78. [10] K. Nag et al., Biophys. J. 71 (1996) 1356^1363. [11] M. Obladen, D. Popp, C. Scholl, H. Schwarz, F. Jahning, Biochim. Biophys. Acta 735 (1983) 215^224. [12] M.A. Launois-Surpas et al., Colloid Polym. Sci. 270 (1992) 901^911. [13] R.C. MacDonald, S.A. Simon, Proc. Natl. Acad. Sci. USA 84 (1987) 4089^4093. [14] I. Panaiotov, T. Ivanova, K. Balashev, J. Proust, Colloids Surf. A Physicochem. Eng. Aspects 102 (1995) 159^165. [15] S.P. Heyn, M. Egger, H.E. Gaub, J. Phys. Chem. 94 (1990) 5073^5078. [16] M. Gugliotti, M.J. Politi, H. Chaimovich, Biochem. Educ. 26 (1998) 233^238. [17] M. Gugliotti, M.J. Politi, H. Chaimovich, J. Colloid Interface Sci. 198 (1998) 1^5. [18] A.M. Carmona Ribeiro, H. Chaimovich, Biochim. Biophys. Acta 733 (1983) 172^179. [19] A.W. Adamson, Physical Chemistry of Surfaces, 5th ed., Wiley, New York, 1990. [20] D.K. Chattoraj, K.S. Birdi, Adsorption and the Gibbs Surface Excess, Plenum Press, New York, 1984. [21] I. Panaiotov, Tz. Ivanova, A.S. Sanfeld, Adv. Colloid Interface Sci. 40 (1992) 147^156. [22] G.A. Lawrie, P.B. Schneider, B.J. Battersby, G.T. Barnes, H.K. Cammenga, Chem. Phys. Lipids 79 (1996) 1^8. [23] S.G. Clerc, T.E. Thompsom, Biophys. J. 68 (1995) 2333^ 2341. [24] C.M. Sabra, K. Jorgensen, O.G. Mouritsen, Biochim. Biophys. Acta 1282 (1996) 85^92. [25] L.F. Gomes, I.M. Cuccovia, H. Chaimovich, D.H.G.P. Barbieri, M.J. Politi, Biochim. Biophys. Acta 1152 (1993) 78^82. [26] A.M. Carmona-Ribeiro, L.S. Yoshida, H. Chaimovich, J. Phys. Chem. 89 (1985) 2928^2933. [27] K. Makino et al., Biophys. Chem. 41 (1991) 175^183. [28] F. Bonosi, G. Gabrielli, Colloids Surf. 52 (1991) 277^285. [29] M. Ca¡rey, J. Hogan, Chem. Phys. Lipids 61 (1992) 1^109. [30] A.G. Lee, Biochim. Biophys. Acta 472 (1977) 237^281. [31] M. Andersson, L. Hammarstro«n, K. Edwards, J. Phys. Chem. 99 (1995) 14531^14538. [32] B.C.R. Guillame, D. Yogev, J.H. Fendler, J. Phys. Chem. 95 (1991) 7489^7494. [33] M. Kodama, T. Kunitake, S. Seki, J. Phys. Chem. 94 (1990) 1550^1554.
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Fusion of vesicles with the air^water interface: the in£uence of polar head group, salt concentration, and vesicle size M. Gugliotti *, H. Chaimovich, M.J. Politi Departamento de Bioqu|¨mica, Instituto de Qu|¨mica, Universidade de Sa¬o Paulo, Av. Prof. Lineu Prestes 748, 05508-940 Sa¬o Paulo, Brazil Received 24 August 1999; received in revised form 12 October 1999; accepted 19 October 1999
Abstract Fusion of vesicles with the air^water interface and consequent monolayer formation has been studied as a function of temperature. Unilamellar vesicles of DMPC, DPPC, and DODAX (X = Cl3 , Br3 ) were injected into a subphase containing NaCl, and the surface pressure (tension) was recorded on a Langmuir Balance (Tensiometer) using the Wilhelmy plate (Ring) method. For the zwitterionic vesicles, plots of the initial surface pressure increase rate (surface tension decrease rate) as a function of temperature show a peak at the phase transition temperature (Tm ) of the vesicles, whereas for ionic ones they show a sharp rise. At high concentrations of NaCl, ionic DODA(Cl) vesicles seem to behave like zwitterionic ones, and the rate of fusion is higher at the Tm . The influence of size was studied comparing large DODA(Cl) vesicles with small sonicated ones, and no significant changes were found regarding the rate of fusion with the air^water interface. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Vesicles; Phase transition temperature; Surface pressure; Fusion; Interface
1. Introduction Vesicles and liposomes may disrupt upon contact with the air^water interface forming a monolayer [1^ 3]. As a consequence of monolayer formation, the surface pressure, Z (or tension, Q) of a vesicle dispersion changes. This phenomenon has been detected in dispersions of vesicles and liposomes made of di¡erent types of lipids, natural and synthetic ones [4,5], and kinetic studies [6^8] have been developed to characterize the process. Fusion (or spreading) of vesicles at the surface of solutions has been used as an alternative to form monolayers with attached pro-
* Corresponding author. Fax: +55-11-815-5579; E-mail: [email protected]
teins [9,10]. Most of the works are focused on the in£uence of temperature on the rate of vesicles fusion with the air^water interface, which has various implications of biological and pharmacological interest, since the transformation of a closed bilayer into a monolayer is an alternative to study the problem of the lung surfactant [11,12]. Fusion of vesicles with the air^water interface is a spontaneous process, and the large in£uence of the phase transition temperature of vesicles (Tm ) [13] on the rate of fusion has been noticed. Fusion becomes much faster as the temperature of the system approaches the Tm [13^15]. The correlation between temperature and rate of fusion leads to the possibility of studying bilayers properties like permeability and interbilayer fusion using this phenomenon, since these properties depend also strongly on the temper-
0005-2736 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 3 6 ( 9 9 ) 0 0 2 2 1 - 7
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ature and are related to structural changes on the membranes. Recently, we showed that is possible to determine the Tm of vesicles using this methodology [16,17]. The aim of the present work is to study the di¡erences between ionic and zwitterionic vesicles regarding the rate of fusion with the air^water interface below and above the Tm . From the results is also possible to estimate de Tm of the vesicles. DODA(Cl) vesicles were chosen to verify the in£uence of salt concentration and size. 2. Materials and methods 2.1. Materials Dioctadecyldimethylammonium chloride and bromide (DODA(Cl)/DODA(Br)) were purchased from Kodak, and recrystallized using methanol/acetone 5:95 (v/v). NaCl (Merck) was of analytical grade. Dimyristoyl/dipalmytoylphosphatidylcholine (DMPC/DPPC) were purchased from Sigma. DPPC and DMPC were analyzed by thin-layer chromatography and showed just one spot. The water used in all experiments was doubly distilled and ¢ltered using a Milli-Q (Millipore) apparatus. 2.2. Methods 2.2.1. Vesicle preparation and properties The concentrations of all vesicle dispersions used are expressed in terms of the lipid monomer concentrations. Di¡erent volumes of small unilamellar vesicles dispersions were prepared by sonicating (15 min) lipid suspensions (4 and 10 mM) at about 10³C above the Tm of the vesicles using the titanium tip sonicator Braunsonic 1510 (B. Braun). The NaCl solutions for sonication were of the same concentration of those used for the surface pressure (tension) assays, in order to ensure an isosmotic medium. After sonication, the vesicle dispersions were centrifuged (10 000Ug for 20 min) in order to remove the titanium particles. Vesicle size was determined by dynamic light scattering (He^Ne laser, 60 mW, EMI photomultiplier, Brookhaven BI90 autocorrelator). Vesicle hydrodynamic diameters were 360 þ 50, î for DODA(Cl), 400 þ 50, 450 þ 50, and 570 þ 50 A DODA(Br), DMPC, and DPPC, respectively. Large
unilamellar DODA(Cl) vesicles were prepared by the injection method [18] yielding vesicles with a diameî . The NaCl concentration for the ter of 3800 þ 50 A light-scattering assays was 1 mM for all types of vesicles. 2.2.2. Surface pressure (Z) and surface tension (Q) measurements The Te£on trough consisted of a hollow circular compartment (diameter 6 cm) connected to a water bath circulator system. The temperature was measured inside the trough using a Te£on-coated thermometer (0.5³C accuracy). Vesicle aliquots were injected into the subphase using a glass pipette, followed by magnetic stirring using a Te£on stir bar. Z was measured by the Wilhelmy plate method [19] on a Langmuir balance (KSV Instruments, model KSV 5000), placed in a clean room. The same procedure was carried out using the Ring method [20], and Q was measured on a Du Nouy tensiometer (Fisher Scienti¢c Tensiomat, Model 21, Fisher Scienti¢c). 2.3. Experimental procedure The assays were carried out in four steps. (1) An aliquot of the vesicle dispersion (1, 3, 5 ml,T) was injected into the subphase. The system was stirred very carefully during the next 10 s. Following this, the surface was cleaned by aspiration using a Te£on tube connected to a vacuum pump. After setting Z to zero, the assay began at the selected temperature; (2) Z was recorded continuously as a function of time; (3) after the measurement, the temperature was set to the new value; (4) when the system reached the new temperature, the surface was cleaned by aspiration and the assay restarted without further stirring. The same procedure was carried out when using the tensiometer, but in this case Q was measured every 5 min. 3. Results 3.1. In£uence of polar head group Fig. 1 shows the results obtained using the Langmuir balance for DODA(Br) and DPPC sonicated vesicles. The surface pressure Z was recorded at sev-
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Fig. 1. (A) vZ/vt as a function of temperature for 5 ml of 5 mM of small DODA(Br) vesicles injected into 35 ml of a 1 mM NaCl subphase. (B) Ten ml of 5 mM of small DPPC vesicles were injected into 30 ml of a 1 mM NaCl subphase. The insets show Z recorded at several temperatures on a Langmuir balance.
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against the temperature, and the same result was found for DODA(Br) vesicles (Fig. 2). For DPPC and DMPC vesicles, vQ/vt showed a peak at 40³C and 25³C, respectively (Fig. 2), which are the Tm for these vesicles. 3.2. In£uence of vesicle size
Fig. 2. vQ/vt as a function of temperature for 7 ml of 5 mM of small DODA(Br) vesicles (F), 5 ml of 2 mM of small DPPC vesicles (a), and 5 ml of 5 mM of small DMPC vesicles (R). The subphases consisted of 40 ml of a 1 mM NaCl solution.
eral temperatures (Fig. 1, insets), and the tangents to the curves taking into account the ¢rst minute (vZ/ vt, where Z is the surface pressure and t is time) were calculated using KSV-LB5000 software, and plotted as a function of temperature. For DODA(Br) vesicles (Fig. 1A) it is possible to note a sharp rise in vZ/vt after 35³C, which corresponds to the Tm of these vesicles. A di¡erent pattern is observed for DPPC vesicles (Fig. 1B), where vZ/vt reaches a maximum at 39³C, and decreases thereafter. The same assay was carried out using the tensiometer for DODA(Br), DPPC, and DMPC sonicated vesicles. The surface tension decrease rate vQ/vt was plotted
Fig. 3. vZ/vt as a function of temperature for 5 ml of 5 mM of small DODA(Cl) vesicles (a), and for 10 ml of 10 mM of large DODA(Cl) vesicles (F) injected into 35 ml of a 1 mM NaCl solution.
Fig. 3 compares the results for small and large unilamellar DODA(Cl) vesicles. From the results obtained using the Langmuir balance, vZ/vt was calculated and plotted against the temperature. The NaCl concentration of the subphase (1 mM) was the same for both types of vesicles. A sharp rise was found at 36³C and 37.5³C for the small and large DODA(Cl) vesicles, respectively. 3.3. In£uence of salt concentration Fig. 4 shows the results of small DODA(Cl) vesicles prepared in 10 mM and 20 mM NaCl solutions. The change on the behavior of the fusion rate was observed in the 10 mM NaCl solution, and for 20 mM of NaCl, the vZ/vt pattern has changed completely. For these two cases, a peak at 40³C determines the Tm , a few degrees higher than at lower concentrations of NaCl. The same result was obtained using a 50 mM NaCl solution as subphase (data not shown).
Fig. 4. vZ/vt as a function of temperature for 10 ml of 5 mM of small DODA(Cl) vesicles injected into 30 ml of NaCl, 10 mM (a) and 20 mM (F).
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Table 1 Comparison between the transition phase temperatures obtained using the tensiometer and the Langmuir balance with the literature values for Tm of sonicated vesicles Vesicles
Tensiometer (³C)
Langmuir balance (³C)
Literature value (³C)
Refs.
DODA(Cl) DMPC DPPC DODA(Br)
^ 25 40 35
36 ^ 39 35
37.5 22^29 41 36^40a
[17,18,28] [29]b [30,31] [31^33]
a
Di¡erent values between 36³C and 40³C are found for DODA(Br) in the literature. Ref. [29] is a database containing 702 records on the Tm of DMPC vesicles. The most common temperature found is 24³C. b
4. Discussion The biggest advantage of this method is that the rate of fusion does not depend on the concentration of the vesicles when high-concentration samples are used [12,14]. According to the results obtained by Pattus and Schindler [2,3], 75% of the vesicles remain like layers directly beneath the water surface in equilibrium with those in the bulk. This equilibrium is governed by the di¡usion of the vesicles to the bulk, which changes when low-concentration samples are used. In addition, the equilibrium between the vesicles is a¡ected during the assay by aspirating the surface, because part of the vesicle population may be lost, decreasing the concentration. Some authors have reported changes on vZ/vt when very low concentrations of vesicles are used [21,22]. Besides, even where using high concentrations of vesicles (5, 10 mM) to avoid this, £uctuations may occur because of the raising and lowering of the ring, when using the tensiometer, due to the change in the equilibrium between the vesicles near the interface and those in the bulk. Thus, for a simple veri¢cation, all assays were repeated at least two times for all types of vesicles, and with several di¡erent total vesicle concentrations, no change in the results was found. A more accurate determination of the Tm by this methodology is possible using lower concentrations of vesicle dispersions. In this case, Z or Q are recorded continuously as a function of temperature, without any interference at the surface (aspiration) during the assay [16,17]. However, in this approach is not possible to detect di¡erences between ionic and zwitterionic vesicles. The in£uence of the polar head group is clearly observed in Figs. 1 and 2. It is interesting to note that, for zwitterionic vesicles, plots
of vZ/vt (or vQ/vt) show similar results to those obtained from permeability studies [23,24], where the maximum in permeability occurs when both the gel and liquid^crystalline phases coexist. The results presented here indicate that the zwitterionic vesicles are relatively stable below and even above the Tm , and at this temperature they fuse with the interface easily. On the other hand, the ionic vesicles showed a continuous increase in the rate of fusion above the Tm . In fact, an increase in permeability of DODA(Cl) vesicles at temperatures above the Tm has been reported [25]. It is known that DODA(X) (X = Cl3 , Br3 ) vesicles are not stable in the presence of high concentrations of salt [26]. Vesicle dispersions prepared with 10 mM of NaCl did not show high turbidity, but the samples prepared with 20 and 50 mM of NaCl were very turbid. However, the addition of salt from 10 up to 50 mM showed the same change on the fusion rate of DODA(Cl) vesicles, that is, a peak instead a sharp rise at the Tm , with a slight increase on its value. This e¡ect is assigned to the lowering of the degree of ionization of the vesicles as a function of the increase in salt concentration in the subphase. As the ionic strength increases, the repulsion between the charged head groups decreases, allowing the lipids to stay closer to each other [27]. This represents a gain in stability, and it is seen as an increase on the Tm . No signi¢cant changes were found between the rate of fusion with the interface for large and small DODA(Cl) vesicles, although di¡erent results are found in the literature [3,6]. It is possible to observe that the sharp rise on vZ/vt occurs at 36³C and 37.5³C for the small and large DODA(Cl) vesicles, respectively, which corresponds to the Tm region for these vesicles.
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Table 1 summarizes the results for Tm determination obtained using the tensiometer and the Langmuir balance for each type of vesicle, and the literature values for Tm are also listed for comparison. The Tm values found by analyzing the rate of fusion are in good agreement with those obtained by other techniques (see references in Table 1). 5. Conclusions The Tm values obtained using the tensiometer and the Langmuir balance are nearly the same, and the results are in good agreement with those obtained by other methods. A di¡erence in the behavior between zwitterionic and ionic vesicles at low salt concentration is observed. The results show that the ionic vesicles continue to fuse with the air^water interface above their Tm , while for the zwitterionic ones the maximum rate of fusion occurs at this temperature. The addition of salt to the ionic vesicles changed their behavior into that of zwitterionic ones. Although DODAX (Cl3 , Br3 ) vesicles do not present good stability in the presence of salt, it was possible to detect the change on the rate of fusion with the air^water interface at the Tm , and a change on its value due to high salt concentrations of NaCl. No signi¢cant changes on the Tm values were found between small and large DODA(Cl) vesicles, since the di¡erence of 1.5³C is within experimental error. These results strongly suggest that the mechanism of fusion with the interface for DODA(Cl) vesicles does not depend on the curvature radii of these bilayers. Acknowledgements The authors wish to thank Fabio Florenzano for the light-scattering measurements and Prof. Dr. Iolanda M. Cuccovia for preparing the injected vesicles. This work was supported by the following Brazilian ¢nancial agencies: CNPq, FAPESP, FINEP, and CAPES. References [1] R. Verger, F. Pattus, Chem. Phys. Lipids 16 (1976) 285^291.
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